uhplc selectivity guide - science unfiltered
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The Chromatographer’s GuideTo Improving UHPLC Column Selectivity
www.phenomenex.com/UHPLC
© 2018 Phenomenex, Inc. All rights reserved.
Understanding Solid Support and Efficiencyp. 3 ................ Selecting the Right Solid Support
p. 4 ................ How Resolution is Affected by Selectivity and Efficiency
p. 5 ................ Efficiency Gains with Luna® Omega and Kinetex®
Step 1
p. 9 ................ Alkyl Phases
p. 10 .............. Phenyl Phases
p. 11 .............. Polar Retentive Phases
Our UHPLC SelectivitiesStep 3
p. 6 ................ Profiling the Mechanisms of Selectivity
p. 7 ................ Relating Selectivity to UHPLC Stationary Phases
pp. 21-23
Step 2How to Use Selectivity
Step 4Applying Column Selectivities to Your UHPLC Analysis
Step 5Order Your New Selectivity Tools
Guide to Choosing the Most Effective Selectivity
pp. 13-15 ....... Thinking Outside the Traditional C18 Phases
pp. 16-17 ....... Improve Polar Separations with 100 % Aqueous Stable Phases
p. 18 .............. Utilizing Unique Selectivities
p. 19 .............. Aromatic Based Selectivitiy
p. 20 .............. Protect your Column’s Selectivity
3
Und
erstanding
So
lid S
upp
ort and
Effi
ciencyStep
1Select the Right Solid SupportPhenomenex offers a full range of solid supports including core-shell and thermally modified fully porous. The morphology of the solid support has a significant impact on the resulting material characteristics and column performance.
Core-Shell and Organo-Silica Core-ShellUnique solid silica core and porous shell that results in faster chromatogra-phy and higher efficiencies than conventional fully porous particles.
Well suited for:
• Performance gains on ANY LC system
• Easy system-to-system and lab-to-lab method transfer
• Methods where increased sensitivity is required
• Significantly improving the productivity of older, established methods
Fully Porous – Thermally Modified SilicaUnique high efficiency and extremely robust fully porous silica that offers astounding performance and inertness alongside versatile selectivities.
Well suited for:
• Astounding UHPLC, HPLC, and Preparative HPLC performance and efficiencies
• Greater separation muscle
• Better peak shape through an inert foundation
• Extreme ruggedness and dependability
Scalability
Capillary Minibore MidBore™ Analytical Semi-Prep Preparative Bulk Media
Particle Sizes
1.3 µm 1.6 µm 1.7 µm 2.5 µm 2.6 µm 3 µm 3.5 µm 4 µm 5 µm 10 µm 15 µm
Scalability
Capillary Minibore MidBore™ Analytical Semi-Prep Preparative Bulk Media
Particle Sizes
1.3 µm 1.6 µm 1.7 µm 2.5 µm 3 µm 3.5 µm 4 µm 5 µm 10 µm 15 µm
Thermal Modified Pore StructureMost importantly, through our proprietary process, we eliminate micropores, further improving column efficiency, inertness, and reproducibility.
Absence of Micropores
Micropores
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4
How Resolution is Affected by Selectivity and EfficiencySelectivity (α) and efficiency (N) have the greatest impact on observed resolution (R), when compared to other chromatographic parameters. Often the simplest and most effective way to improve your chromatographic results is to change your column’s phase or solid support. Phenomenex offers a wide breadth of phase chem-istries across multiple solid supports for simplified method development and optimization.
The Impact of Selectivity on Resolution
0
0 5 10 15 20 25
Ν
Ν
α
α
4R = Να
α+1
-1 ( )( )( )
0
0.5
1.0
1.5
Res
olut
ion
(R)
2.0
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3.0
1.00 1.05 1.10 1.15 1.20 1.25
5,000 10,000 15,000 20,000 25,000
Retention Selectivity Efficiency
Retention
Selectivity
Efficiency
Selectivity is the most important parameter for increasing resolution.
Step1
Und
erst
and
ing
So
lid S
upp
ort
and
Effi
cien
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5
The undeniably high efficiency levels found in each Luna® Omega and Kinetex® column provide you with the potential of huge gains in method performance. While traditional silica and hybrid fully porous particles may claim high performance, when compared to Luna Omega or Kinetex, they fall short and prevent HPLC/UHPLC scientists from reaching their goals.
Step1
Comparative separations may not be representative of all applications.
Efficiency Levels (plates/m)
Kinetex 1.3 µm
Luna Omega 1.6 µm
Kinetex 1.7 µm
Conventional Fully Porous 1.7 µm
Kinetex 2.6 µm
100,000 200,000 300,000 400,000
5 10 15 20 25 min0
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Ap
p ID
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Ap
p ID
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p ID
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UHPLC Performance – Cyclosporine Impurity Profile
Conditions for all columns same except where noted:Columns: Kinetex 2.6 μm Polar C18
Luna Omega 1.6 μm Polar C18Conventional Fully Porous 1.7 μm C18
Dimensions: 50 x 2.1 mmMobile Phase: Acetonitrile/Tert-butyl methyl ether/Water/
Phosphoric acid (430:50:520:1)Flow Rate: 0.30 mL/min
Temperature: 80 ˚CDetection: UV @ 210 nm
Sample: Cyclosporine
Kinetex 2.6 μm Polar C18
Conventional 1.7 μm C18
Luna Omega 1.6 μm Polar C18
Pressure: 70 bar
Pressure: 126 bar
Pressure: 117 bar
Valuable Resolution Valuable Resolution
Disappointing Resolution
Efficiency Gains with Luna Omega and KinetexU
nderstand
ing S
olid
Sup
po
rt and E
fficiency
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6
Profiling the Mechanisms of SelectivityObserved selectivity is dictated by several primary molecular interactions. Below you’ll find selectivity parameters used to help characterize reversed phase selectivity mechanisms.
HydrophobicityThe ability of a phase to
hydrophobically interact with carbon groups
Steric InteractionThe ability of a phase to
separate compounds based on structural differences
Cation Selectivity at pH 2.8The ability of a phase to interact with
cation groups at acidic pH
Cation Selectivity at pH 7.0The ability of a phase to interact with
cation groups at neutral pH
O O
B+
O–
O O
C π - ππ - π
Hydrogen Bond Donating Capacity
The ability of a phase to hydrogen-bond with proton
accepting groups
O OO
H
B∙∙
Hydrogen Bond Accepting CapacityThe ability of a phase to
hydrogen-bond with proton donating groups
O O∙∙X
OH
Ho
w t
o U
se S
elec
tivi
tyStep
2
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NH2HO
HO
Relating Selectivity to UHPLC Stationary PhasesFollow the colors to connect functional groups to selectivity profiles! This color coordination demonstrates the relationship between atomic fragments of compounds and how to relate them to column selectivity profiles.
Hydrophobicity
π-π Interaction
O O
C
O O∙∙X
OH
π - ππ - π
Example: Luna® Omega 1.6 µm Polar C18 Selectivity Bar Chart
Hydrophobicity
Steric Interaction
Hydrogen Bond Donating Capacity
Hydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8
Cation Selectivity at pH 7.0
Low High
Hydrogen Bond Accepting
Capacity
Define the Characteristics of Your Target Compounds
Match Your Target Compounds to the 5 Primary Selectivity ParametersThree primary mechanisms of selectivity relate to the analyte of interest above. By referencing the column selectivity bar chart below, we can see the correlation between selectivity mechanisms and column selectivity profiles.
Step2
Ho
w to
Use S
electivity
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8
p. 9 Alkyl PhasesUHPLC selectivities that are best suited for the analysis of hydrocarbons
p. 10 Phenyl PhasesUHPLC selectivities that are best suited for aromatic compounds
p. 11 Polar Retentive PhasesUHPLC selectivities that are best suited for more polar compounds
Selectivity OverviewO
ur U
HP
LC S
elec
tivi
ties
Step3
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9
Alkyl Phases
Application Spotlight• Cannabinoids
• Analgesics
• Pharmaceuticals (USP: L1)
Find the right amount of hydrophobic selectivity for your separa-tion. Below are UHPLC columns that are recommended for the separation of hydrocarbon-based compounds.
Material Characteristics
PhaseParticle
Sizes (µm)Pore Size
(Å)Effective Surface
Area (m²/g)Effective
Carbon Load %pH
StabilityPressure Stability
Luna Omega C18 1.6 100 260 11 1.5 - 8.5*
1,000/600† bar
Kinetex XB-C18 1.7, 2.6, 3.5, 5 100 200 10 1.5-8.5*
Kinetex C18 1.3, 1.7, 2.6, 5 100 200 12 1.5-8.5*
Kinetex C8 1.7, 2.6, 5 100 200 8 1.5-8.5*
Kinetex EVO C18 1.7, 2.6, 5 100 200 11 1.0-12.0
* pH stability under gradient conditions. pH stability is 1.5 - 10 under isocratic conditions.† 2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.
When using Kinetex 1.3 µm or 1.7 µm, increased performance can be achieved, however high pressure-capable instrumentation is required.
USP: L1
TMSTMS
Luna® Omega C18Rugged and highly efficient C18 with strong focus on hydrophobic retention of non-polar and polar compounds.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L1
Si TMSTMS
Kinetex® XB-C18Di-isobutyl side chains differentiate this C18 column. Low ligand density and an inactive surface make this column a great hydrogen acceptor. This phase will demonstrate improved peak shape for basic compounds and increased retention of acids.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L1
TMSTMS
Kinetex C18Very well balanced column providing some selectivity through steric, hydrogen, and cationic pathways. This is a great starting point for ultra-high efficiency separations.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L7
TMSTMS
Kinetex C8Brings the benefits of core-shell technology to USP L7 methods. The phase will provide moderate hydrophobicity and good steric and hydrogen donating selectivity.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L1
TMSTMS Kinetex EVO C18Novel pH 1-12 stable C18 that delivers robust methods and improved peak shape for bases.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
O O
HydrophobicityHigh column hydrophobicity values indicate greater retention of carbon-
containing analytes.
Denotes stationary phases that are 100 % aqueous stable
Our U
HP
LC S
electivitiesStep
3
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10
Application Spotlight• Taxanes
• Mycotoxins
• Opiates
Aromatic interactions greatly promote steric interactions. Below are UHPLC columns that have the highest potential for pi-pi bond interaction.
Phenyl Phases
USP: L11
TMSTMS Kinetex® Biphenyl100 % aqueous stable reversed phase chemistry with hydrophobic‚ aromatic‚ and enhanced polar selectivity.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L11
TMSTMS
Kinetex Phenyl-HexylAromatic and moderate hydrophobic selectivity result in the great retention and separation of aromatic hydrocarbons.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L43
F
F
F
F
F
TMSTMS
Kinetex F5This pentafluorophenyl propyl column provides a veryhigh degree of steric selectivity to separate structural isomers. The electronegative fluorine groups offer high selectivity for cationic compounds.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
Material Characteristics
PhaseParticle
Sizes (µm)Pore Size
(Å)Effective Surface
Area (m²/g)Effective
Carbon Load %pH
StabilityPressure Stability
Kinetex Biphenyl 1.7, 2.6, 5 100 200 11 1.5-8.5*
1,000/600† barKinetex Phenyl-Hexyl 1.7, 2.6, 5 100 200 11 1.5-8.5*
Kinetex F5 1.7, 2.6, 5 100 200 9 1.5-8.5
* pH stability under gradient conditions. pH stability is 1.5 - 10 under isocratic conditions.† 2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.
When using Kinetex 1.3 µm or 1.7 µm, increased performance can be achieved, however high pressure-capable instrumentation is required.
π - ππ - π
Try using methanol for the organic portion of the mobile phase. It can help promote pi-pi bond interaction!
Method Develoment Tip!
Denotes stationary phases that are 100 % aqueous stable
AromaticityColumn chemistries that contain ring structures interact with aromatic or
phenyl containing compounds via pi-pi interactions (π stacking).
Our
UH
PLC
Sel
ecti
viti
esStep
3
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11
Application Spotlight• Peptide Mapping
• Pesticides
• Nucleosides
Column phases with hydrogen bond accepting/donating capacity have increased polar selectivity and retention of polar compounds. Below are UHPLC columns that offer increased polar selectivity/retention.
Polar Retentive Phases Hydrogen Bond Accepting Capacity
Hydrogen bond accepting groups on the silica surface interact with hydrogen
bond donating functionalities on analytes.
O O∙∙X
OH
USP: L1
TMSTMS
PolarPolar
Luna® Omega Polar C18100 % aqueous stability and enhanced selectivity/retention for polar analytes without diminishing useful non-polar retention. The C18 ligand provides general hydrophobic interactions while a polar modified particle surface provides enhanced polar compound retention.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L1
TMSTMS
++
Luna Omega PS C18Unique, 100 % aqueous stable mixed-mode phase that provides both polar and non-polar retention. The surface contains a positive charged ligand which aids in the retention of acidic compounds through ionic interactions, while the C18 ligand promotes general reversed phase hydrophobic retention. The positively charged surface also improves basic compound peak shape through ionic repulsion.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
USP: L1
TMSTMS
PolarPolar
Kinetex® Polar C18Combined C18 and polar modified surface that provide polar and non-polar retention alongside 100 % aqueous stability.
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
Material Characteristics
PhaseParticle Sizes
(μm)Pore Size
(Å)Surface Area
(m²/g)Carbon Load
(%)pH
StabilityPressure Stability
Kinetex Polar C18 2.6 100 200 9 1.5 - 8.5*
1,000/600† barLuna Omega Polar C18 1.6, 3, 5 100 260 9 1.5 - 8.5*
Luna Omega PS C18 1.6, 3, 5 100 260 9 1.5 - 8.5*
* pH stability under gradient conditions. pH stability is 1.5 - 10.0 under isocratic conditions.† 2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.
Denotes stationary phases that are 100 % aqueous stable
Our U
HP
LC S
electivitiesStep
3
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12
Applying Column Selectivities to Your UHPLC Analysis
pp. 13-15 Thinking Outside the Traditional C18 PhasesAdvances over traditional C18 selectivity
pp. 16-17 Improve Polar Separations with 100 % Aqueous Stable PhasesThe selectivity power of using aqueous stable UHPLC columns
p. 18 Utilizing Unique SelectivitiesComparison of unique stationary phases
p. 19 Aromatic Based SelectivityAromatic (pi-pi interaction)
p. 20 Protect the Column’s SelectivityExtend your column’s lifetime
Browse thousands of applications at:www.phenomenex.com/applications
™
Ap
ply
ing
Sel
ecti
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Step4
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2, 4
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0.50 1 1.5 2 2.5 3 3.5 min0
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1.2e5
1.3e5
1.4e5
1.5e5
24613
The extent to which resolution depends on selectivity becomes evident when a chromatogra-pher takes advantage of technologic advances in polar stationary phases in order to improve their analysis. Below is an example of resolution improvements obtained by switching from a traditional C18 to a column with additional selectivity mechanisms.
Polar Selectivity of Catecholamines
Inte
nsity
, cp
sIn
tens
ity, c
ps
Luna® Omega 1.6 μm Polar C18
Kinetex® 1.7 µm C18
Ap
p ID
246
09A
pp
ID 2
4611
Comparative separations may not be representative of all applications.
Polar Modified
Traditional C18
Ap
plying
Selectivity
Step4
USP: L1
TMSTMS
PolarPolar
HILIC
TMSTMS
Conditions for both columns:Columns: Luna Omega 1.6 µm Polar C18
Kinetex 1.7 µm C18Dimensions: 50 x 2.1 mm
Mobile Phase: A: 10 mM Ammonium Formate with 0.1 % Formic Acid B: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min)03
% B090
Flow Rate: 0.4 mL/minInjection Volume: 1 µL
Temperature: 22 °CDetection: MS/MS (SCIEX API 4000™)
Sample: 1. Norepinephrine2. Epinephrine3. Normetanephrine4. Dopamine5. Metanephrine6. Serotonin
Improved Selectivity for Polar Analytes
Thinking Outside the Traditional C18 Phase
HO NH2
HODopamine
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
Improved Separation with Polar C18 The increased hydrogen bond accepting capacity of the Luna Omega Polar C18 column improves polar selectivity for analytes such as dopamine.
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14
Thinking Outside the Traditional C18 PhaseTraditional C18 phases may not always be the best option. A UHPLC column that has both polar and hydrophobic versatility allows for great method devel-opment flexibility.
Comparative separations may not be representative of all applications.
0 1
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45
6
7
2 3 4 5 min
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0.15
0.20
0.25
0.30
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0.55
23714
AU
Waters® ACQUITY® BEH 1.7 μm C18
0 1
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0.10
0.15
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0.30
0.35
0.40
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23707
Ap
p ID
237
14A
pp
ID 2
3707
AU
Conditions for both columns:Columns: Luna Omega 1.6 μm PS C18
ACQUITY BEH 1.7 μm C18Dimensions: 50 x 2.1 mm
Mobile Phase: A: Water with 0.1 % Formic AcidB: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min)055.018
% B59555
Flow Rate: 0.4 mL/minTemperature: 22 °C
Detection: UV @ 254 nmSample: 1. Uracil
2. Pindolol3. Chlorpheniramine4. Nortriptyline5. 3-Methyl-4-nitrobenzoic acid6. 5-Methyl salicylaldehyde7. Hexanophenone
Better Resolution and Peak Shape
Luna Omega 1.6 μm PS C18
Acids/Bases/Neutrals
PositiveSurface Charge
Traditional C18
HILIC
TMSTMS
USP: L1
TMSTMS
++
Ap
ply
ing
Sel
ecti
vity
Step4
Improved Separation with PS C18 The increase in steric interaction of the Luna Omega PS C18 column improves resolution for aromatic analytes such as nortriptyline.
NH
Nortriptyline
HydrophobicitySteric Interaction
Hydrogen Bond Donating CapacityHydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8Cation Selectivity at pH 7.0
Low High
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Traditional UHPLC C18 phases can be prone to peak tailing for highly basic compounds and this tailing can become exacerbated when higher loadability is required. The Luna® Omega PS C18’s combination of a high surface area and novel surface chemistry allows for better peak shape as loading increases.
Thinking Outside the Traditional C18 Phase
0.00
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AU
0
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Amitriptyline Loading Study
Conditions for both columns:Columns: Luna Omega 1.6 μm PS C18
ACQUITY BEH 1.7 μm C18Dimensions: 50 x 2.1 mm
Mobile Phase: A: Water with 0.1 % Formic AcidB: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min)05
% B580
Flow Rate: 0.4 mL/minTemperature: 22 °C
Detection: UV @ 254 nmSample: Amitriptyline
AU
Better Peak Shape Over All Loads
Waters® ACQUITY® BEH 1.7 μm C18
Luna® Omega 1.6 µm PS C18
Luna Omega PS C18 50 x 2.1 mm vs. Other Columns
Pea
k W
idth
at
50 %
0 0.1 0.2 0.3 0.4 0.5 0.6
0
0.09
0.08
0.07
0.05
0.06
0.04
0.03
0.02
0.01
Luna 1.6 µm PS C18
Advanced Materials Technology HALO® 2 µm C18
Waters ACQUITY® BEH 1.7 µm C18
Thermo Hypersil GOLD® 1.9 µm C18
Ap
p ID
237
39
Better Peak Shape Over All Sample Loadings
Comparative separations may not be representative of all applications.
Ap
p ID
237
38A
pp
ID 2
3737
Positive Surface Charge
Traditional C18
HILIC
TMSTMS
USP: L1
TMSTMS
++
Ap
plying
Selectivity
Step4
Why is Loadability Important? • Analytical conformation parallel to purification
• Dealing with high API concentrations when conducting stability tests
• High sample loading to visualize low-level analytes of interest
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16
Inte
nsity
, cp
sIn
tens
ity, c
ps
Improve Polar Separations with 100 % Aqueous Stable PhasesA powerful tool in the chromatographer’s toolbox is the ability to use 100 % aqueous conditions to promote polar selectivity and increased retention. Traditional C18 phases are known to collapse under 100 % aqueous conditions, causing a loss of retention and method development headaches.
Catecholamines
0.20 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min0.0
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0.20 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min
Ap
p ID
244
10A
pp
ID 2
4408
Luna® Omega 1.6 µm Polar C18
Luna® Omega 1.6 µm Polar C18
Conditions for both separations:
Column: Luna Omega 1.6 µm Polar C18Dimension: 50 x 2.1 mm
Part No.: 00B-4748-ANMobile Phase: A: Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic AcidGradient: Time (min)
03
% B3 (except where noted)100
Flow Rate: 0.4 mL/min
Temperature: 40 °CDetection: MS/MS (SCIEX API 4000™) (ambient)
Sample: 1. Norepinephaine2. Epinephrine3. Normetanephrine4. Dopamine5. Metanephrine6. 3-Hydroxytyramine
USP: L1
TMSTMS
PolarPolar
USP: L1
TMSTMS
PolarPolar
Greater Retention and Resolution Under 100 % Aqueous Conditions
3 % Initial Organic Gradient
100 % Aqueous Organic Gradient
Ap
ply
ing
Sel
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Step4
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1.0e6
2.0e6
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67
1
02.0e54.0e56.0e58.0e51.0e61.2e61.4e61.6e61.8e62.0e6
2
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3
4
4
56
7
min1
1
Vitamin Tablet
Improve Polar Separations with 100 % Aqueous Stable PhasesDipeptides in 100 % Aqueous Conditions
Column: Luna Omega 1.6 µm Polar C18Dimensions: 50 x 2.1 mm
Part No.: 00B-4748-ANMobile Phase: A: Water with 0.1 % TFA
B: Acetonitrile with 0.1 % TFAGradient: Time (min) % B
0 03 75
Flow Rate: 0.6 mL/minInjection Volume: 1 µL
Temperature: 40 °CDetection: UV @ 210 nm (ambient)
Sample: 1. Arg-Glu 2. Gly-Tyr3. Trp-Gly4. Gly-Trp5. Pro-Trp
Column: Competitor 1.7 µm C18Dimensions: 50 x 2.1 mm
Mobile Phase: A: Water with 0.1 % TFAB: Acetonitrile with 0.1 % TFA
Gradient: Time (min) % B0 33 75
Flow Rate: 0.6 mL/minInjection Volume: 1 µL
Temperature: 40 °CDetection: UV @ 210 nm (ambient)
Sample: 1. Arg-Glu 2. Gly-Tyr3. Trp-Gly4. Gly-Trp5. Pro-Trp
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min
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1250
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1750
mAU
Ap
p ID
246
12A
pp
ID 2
4613
Traditional Competitor 1.7 µm C18
Conditions same for both separations:
Column: Luna Omega 1.6 µm Polar C18Dimensions: 50 x 2.1 mm
Part No.: 00B-4748-ANMobile Phase: A: 10 mM Ammonium Formate with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic AcidGradient: Time (min)
044.17
% B09000
Flow Rate: 0.4 mL/minTemperature: 40 °C
Detection: MS/MS (SCIEX API 4000™) @ 450 °CSample: 1. Pyridoxamine
2. Thiamine3. Nicotinic acid4. Pyridoxine
5. Pantothenic acid6. Folic acid7. Riboflavin
Inte
nsity
, cp
s
Ap
p ID
235
79
Inte
nsity
, cp
s
Ap
p ID
235
84
Standards
Water Soluble Vitamins under 100 % Aqueous Conditions
Comparative separations may not be representative of all applications.
Luna® Omega 1.6 µm Polar C18
100 % Aqueous Conditions Increase Retention and Selectivity of Polar Analytes
Ap
plying
Selectivity
Step4
Phenomenex l WEB: www.phenomenex.com
18
23445
1 2 3 4 min0
10 2 3 4 min
23443
Utilizing Unique SelectivitiesThe elution order of analytes can change depending upon the predominate selectivity characteris-tics of a UHPLC column and the utilized mobile phase. Therefore, by altering the stationary phase or mobile phase conditions, we can observe a unique elution order and extend retention of more polar analytes.
Conditions for both columns:Columns: Luna Omega 1.6 µm C18
Kinetex 1.7 µm BiphenylDimensions: 50 x 2.1 mm
Part No.: 00B-4742-AN00B-4628-AN
Mobile Phase: A: Water with 0.1 % Formic AcidB: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min)0455.1
% B595955
Flow Rate: 0.4 mL/minTemperature: 40 °C
Detection: MS/MS (SCIEX API 4000™) (ambient)Sample: Drugs of Abuse
Luna® Omega 1.6 µm C18
Kinetex® 1.7 µm Biphenyl
Ap
p ID
234
43
pH 2.5
pH 2.5
Strong hydrophobic retention and steric interaction
Increased aromatic selectivity and polar retention
Ap
p ID
234
45
USP: L11
TMSTMS
USP: L1
TMSTMS
Ap
ply
ing
Sel
ecti
vity
Step4
Phenomenex l WEB: www.phenomenex.com
Ap
p ID
225
84
0
02.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
1 2 3 40.5 1.5 2.5 3.5 4.5 min
Inte
nsity
, cp
s
19
Increased Separation of Aromatic Compounds Using a Biphenyl Phase
Kinetex 1.7 µm C18 Kinetex 1.7 µm Biphenyl
Aromatic Based SelectivityCompounds with aromatic ring structures offer a specific type of selectivity associated with pi-pi bond interaction. The compound’s aromaticity provides pi electrons that have the potential to interact with pi bonds, which can be found on phenyl-based stationary phases. This provides a unique, orthogonal selectivity compared to a traditional C18 phase.
Column: Kinetex® 1.7 µm BiphenylDimensions: 50 x 2.1 mm
Part No.: 00B-4628-ANMobile Phase: A: Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic AcidGradient: Time (min) % B
0 54 1005 1007 5
Flow Rate: 0.5 mL/minInjection Volume: 10 µL
Temperature: 40 °CDetection: MS/MS (SCIEX API 4000™) (ambient)
Sample: Drugs of Abuse
Conditions for both columns:Columns: Kinetex® 1.7 µm C18
Kinetex® 1.7 µm BiphenylDimensions: 50 x 2.1 mm
Part No.: 00B-4628-AN00B-4475-AN
Mobile Phase: A: Water with 0.1 % Formic AcidB: Methanol with 0.1 % Formic Acid
Gradient: Time (min)044.1
% B1010010
Flow Rate: 400 µL/minInjection Volume: 10 µL
Temperature: 50 °CBackpressure: 450 Bar
Detection: MS/MS (SCIEX API 4000™) (ambient)Sample: 1. Morphine
2. Hydromorphone
Inte
nsity
, cp
s
Inte
nsity
, cp
s
CH3
NH3
O
O
NH3
CH3
CH3O
O
HNO
O
N
H3CO
N
N
Cl
CH3
H3CO
HHO
O
N
H3C NN
N
N
Cl
Ap
plying
Selectivity
Step4
0.50 1.0 1.5 min0
1.0e4
2.0e4
3.0e4
4.0e4
4.4e41
2
0.50 1.0 1.5 min0
1.0e4
2.0e4
3.0e4
4.0e4
4.8e4 1
2
Ap
p ID
246
37
Ap
p ID
246
38
Greater Separation Power
Phenomenex l WEB: www.phenomenex.com
20
ULTRAUHPLC Column Protection
Protect Your Column’s Selectivity
Save Time and MoneyIt’s a fact! Chemical contaminants and particulates are a natural part of any chromatographic analysis. The easiest way to extend column performance is to remove these contaminants and particulates with SecurityGuard ULTRA before they reach your UHPLC column and degrade your chromatography.
With SecurityGuard ULTRA, you will experience:• Increased UHPLC column lifetime
• Better column performance
• More reproducible chromatography
• Fewer wasted columns
With SecurityGuard ULTRA
Without SecurityGuard ULTRA
(24000 times magnification)
(24000 times magnification)
Inlet Frit
No contaminant or particle buildup
Column Media
Intact media
Contaminant and particle buildup
Column Media
Crushed media and fines
Inlet Frit
SecurityGuard ULTRAFor all core-shell and/or < 3 μm particle columns (< 20,000 psi / 1,378 bar)
We used to have to change out our columns every 2 to 3 months and ever since we started using the SecurityGuard cartridges we can do at least 6 months before changing a column out.
T. Serviss
The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.
Ap
ply
ing
Sel
ecti
vity
Step4
Cartridge Holder
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21
Chat Now! www.phenomenex.com/chat
A Phenomenex Technical Specialist is here to help nearly 24 hours a day!
You have things to do.How can we help?
Ap
plying
Selectivity
Step4
Phenomenex l WEB: www.phenomenex.com
22
Kinetex Core-Shell UHPLC Column Ordering Information
1.7 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pkEVO C18 –– 00B-4726-AN 00D-4726-AN 00F-4726-AN AJ0-9298
F5 –– 00B-4722-AN 00D-4722-AN 00F-4722-AN AJ0-9322
Biphenyl –– 00B-4628-AN 00D-4628-AN 00F-4628-AN AJ0-9209
XB-C18 00A-4498-AN 00B-4498-AN 00D-4498-AN 00F-4498-AN AJ0-8782
C18 00A-4475-AN 00B-4475-AN 00D-4475-AN 00F-4475-AN AJ0-8782
C8 00A-4499-AN 00B-4499-AN 00D-4499-AN 00F-4499-AN AJ0-8784
HILIC 00A-4474-AN 00B-4474-AN 00D-4474-AN –– AJ0-8786
Phenyl-Hexyl –– 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788
for 2.1 mm ID
1.7 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 100 x 3.0 3/pkXB-C18 00A-4498-Y0 00B-4498-Y0 00D-4498-Y0 AJ0-8775
C18 –– 00B-4475-Y0 00D-4475-Y0 AJ0-8775
C8 00A-4499-Y0 00B-4499-Y0 00D-4499-Y0 AJ0-8777
HILIC –– 00B-4474-Y0 –– AJ0-8779
for 3.0 mm ID
1.7 μm Microbore Columns (mm) 1.3 μm Minibore Columns (mm)Phases 50 x 1.0 100 x 1.0 150 x 1.0 Phases 30 x 2.1 50 x 2.1EVO C18 00B-4726-A0 00D-4726-A0 00F-4726-A0 C18 00A-4515-AN 00B-4515-AN
Biphenyl 00B-4628-A0 00D-4628-A0 00F-4628-A0
Core-Shell Technology
GlycosBIO® Food SciencesI really love the Kinetex columns. I now have shorter HPLC
method runs and nice peak resolution. Methods that used
to take 30 to 40 minutes on other columns now take 15−20
minutes. I’m enjoying the fact that my samples are analyzed
more quickly without compromising the quality of the
peaks in the chromatograms.
Amgen®
The Kinetex column has worked great for our validated
assays. We easily converted our HPLC methods to UPLC
methods using the Kinetex column and have enjoyed being
able to run fast UPLC chromatography...
University of Texas MD Anderson Cancer CenterWe have drastically improved sensitivity, reproducibility,
and lifetime on our column after switching to
the Kinetex technology.
As Voted by YouKinetex® Core-Shell LC Columns Earned the SelectScience Gold Seal of Quality Award
Ord
erin
g In
form
atio
nStep
5
‡SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000
The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.
Phenomenex l WEB: www.phenomenex.com
23
1.6 μm Microbore Columns (mm)Phases 50 x 1.0 100 x 1.0 150 x 1.0Polar C18 00B-4748-A0 00D-4748-A0 00F-4748-A0
C18 00B-4742-A0 00D-4742-A0 00F-4742-A0
PS C18 00B-4752-A0 00D-4752-A0 -
1.6 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pkPolar C18 00A-4748-AN 00B-4748-AN 00D-4748-AN 00F-4748-AN AJ0-9505
PS C18 00A-4752-AN 00B-4752-AN 00D-4752-AN 00F-4752-AN AJ0-9508
C18 00A-4742-AN 00B-4742-AN 00D-4742-AN 00F-4742-AN AJ0-9502
for 2.1 mm ID
Luna Omega UHPLC Column Ordering Information
OMEGA
Luna® Omega Offers Better UHPLC Column LifetimesPhenomenex columns are engineered for durability and are able to withstand high system pressure. For example, lifetime studies are conducted to ensure superior performance and long column lifetimes.
Accelerated Lifetime Study
% In
crea
se o
ver
Initi
al B
ackp
ress
ure
Injection Cycle
0 10 20 30 40 50 600%
1%
2%
3%
4%
5%
6%
7%
8%
9%
10%
Conditions for both columns:Columns: Luna Omega 1.6 µm C18
ACQUITY BEH 1.7 µm C18Dimensions: 50 x 2.1 mm
Mobile Phase: A: Water with 0.1 % Formic AcidB: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min)044.1
% B5955
Flow Rate: 0.4 mL/minTemperature: 25 °C
Detection: UV @ 210 nmSample: Protein Matrix
Luna® Omega 1.6 µm C18 (AVG)
Waters® ACQUITY® BEH 1.7 µm C18 (AVG)
Less Increase in Pressure Over Time
Ord
ering Info
rmatio
nStep
5
‡SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000
Ap
p ID
236
10
Phenomenex l WEB: www.phenomenex.com
If Phenomenex analytical columns do not provide at least an equivalent separation as compared to a competing column of the same particle size, similar phase and dimensions, return the Phenomenex column with comparative data within 45 days for a FULL REFUND.
The Chromatographer’s Guide
To Improving UHPLC Column Selectivity
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Terms and Conditions Subject to Phenomenex Standard Terms & Conditions, which may be viewed at www. phenomenex.com/TermsAndConditions.Trademarks Luna and Kinetex are registered trademarks and MidBore and SecurityGuard are trademarks of Phenomenex. Waters and ACQUITY are registered trademarks of Waters Technologies Corporation. HALO is a registered trademark of Advanced Materials Technology, Inc. Hypersil GOLD is a registered trademark of Thermo Hypersil-Keystone LLC. API 4000 is a trademark of AB SCIEX Ltd. AB SCIEX ™ is being used under license. SelectScience is a registered trademark of SelectScience LLC.Disclaimer Phenomenex is not affiliated with Advanced Materials Technology, Thermo, or Waters. Comparative separations may not be representative of all applications.Kinetex EVO is patented by Phenomenex. U.S. Patent Nos. 7,563,367 and 8,658,038 and foreign counterparts.The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.© 2018 Phenomenex, Inc. All rights reserved.
www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at international@phenomenex.com
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